Project Summary/Abstract Many placenta-associated pregnancy complications, including pre-eclampsia (PE) and intrauterine growth restriction (IUGR), originate in the early stages of placental development, during the establishment of trophoblast cells, the functional building blocks of the placenta. Due to the inaccessibility of this early stage of human development, most of what we know about this process comes from other species, in particular, from mice. Trophoblast specification in pre-implantation mouse embryos is dependent upon the Tead4/Yap1 complex initiating a trophoblast-specific transcriptional program. However, an increasing body of evidence, from our lab and others?, shows significant species-specific differences in trophoblast lineage specification between mouse and human. In a recent comparative placentation study, we identified the transcriptional co-factor vestigial-like family member-1 (VGLL1) as a human-specific marker of early gestation cytotrophoblast (CTB), the stem cell compartment of the human placenta. VGLL1 co-localizes with TEAD4 and TP63 in early gestation CTB. Furthermore, expression of VGLL1 is induced very early during trophoblast differentiation of human pluripotent stem cells (hPSCs) following BMP4 treatment, before induction of TP63. Little is known about VGLL1 function, but interestingly, VGLL1 has been shown to bind TEAD4, in a manner similar to the Tead4/Yap1 interaction, which is required for trophoblast lineage specification in mice. This project aims to elucidate the role of VGLL1 as a human-specific regulator of trophoblast lineage specification. We will manipulate VGLL1 expression in both newly- established human trophoblast stem cell lines and in our established hPSC-based model of human trophoblast lineage specification. We will assess the effects of such gene manipulation on trophoblast maintenance, differentiation and function, as well as genome-wide changes in RNA expression. We will validate our results by manipulation of VGLL1 in human primary CTB using the CRISPR/Cas9 technology. We will confirm the interaction between VGLL1 and TEAD4 in both systems and identify direct downstream targets of the VGLL1-TEAD4 complex using ChIP-seq. We hypothesize that VGLL1 is a key player involved in trophoblast lineage specification in early human development, acting in complex with TEAD4, to induce a TP63-based transcriptional program to establish and maintain trophoblast stem cells in the human placenta.